Download SialylTn-mAb17-1A Carbohydrate−Protein Conjugate Vaccine

Bioconjugate Chem. 2005, 16, 1519−1528
1519
SialylTn-mAb17-1A Carbohydrate-Protein Conjugate Vaccine:
Effect of Coupling Density and Presentation of SialylTn
Ralf Kircheis,* Petra Vondru, Andreas Nechansky, Renate Ohler, Hans Loibner,
Gottfried Himmler, and Geert C. Mudde
igeneon AG, Immunotherapy of Cancer, Vienna, Austria. Received June 1, 2005
Carbohydrate antigens resulting from aberrant glycosylation of tumor cells, such as SialylTn, represent
attractive targets for cancer vaccination. However, T-cell-independent carbohydrate antigens are poorly
immunogenic and fail to induce memory and IgG class switch. Clustered expression patterns of some
carbohydrates on the cell surface add further complexity to the design of carbohydrate-based vaccines.
We describe here a vaccine consisting of SialylTn carbohydrate epitopes coupled to a highly
immunogenic carrier molecule, mAb17-1A, adsorbed on alhydrogel and coformulated with a strong
adjuvant, QS-21. The SialylTn-mAb17-1A conjugate vaccine was administered in Rhesus monkeys,
and the immune responses against mAb17-1A, SialylTn, ovine submaxillary mucin, and tumor cells
were analyzed. The data demonstrate that the density of carbohydrate epitopes on the carrier is an
essential parameter for induction of anti-carbohydrate specific memory IgG immune responses.
Furthermore, the influence of different types of presentation of SialylTn (monomeric vs trimers vs
clustered via a branched polyethylenimine linker) on antibody titers and specificity was studied. Highdensity coupling of SialylTn epitopes to mAb17-1A induced the strongest immune response against
synthetic SialylTn and showed also the highest reactivity against natural targets, such as OSM and
tumor cells.
INTRODUCTION
Accumulating data indicate that tumor-recognizing
antibodies and immune effector cells may play a deciding
role for the long-term benefit of cancer therapy. Elimination of circulating tumor cells and eradication of micrometastases which remain after surgery or radiotherapy are considered primary targets for immune
cancer therapy. Therefore, identification and selection of
the appropriate target antigen(s) on the tumor cells is
essential for therapy efficacy. Cell surface-exposed carbohydrate and mucin antigens resulting from aberrant
glycosylation of tumor cells may provide attractive targets. In animal models (1-3) as well as cancer patients
the presence of natural or vaccine-induced antibodies
against carbohydrates such as GM2 and SialylTn was
found to correlate with prolonged survival (4-6). The
mucin-derived SialylTn is expressed in more than 80%
of cancers of breast, colorectal, prostate, and ovarian
origin showing no or very limited expression on the
corresponding normal tissues (7-9). SialylTn expression
by various epithelial cancers correlates with a more
aggressive phenotype and poor prognosis (10, 11). Recent
data indicate that mucin-derived O-glycosylated truncated carbohydrates, such as the monosaccharide Tn or
the disaccharides TF or SialylTn, are predominantly
expressed as molecular clusters on the surface of tumor
cells (9, 12-17). The exact pattern of these clusters is
still under debate; clustering in trimers has been suggested as one favorable pattern for antibody recognition
found on ovine submaxillary mucin (OSM) and on the
surface of some tumor cells (14-17). However, it is rather
* Correspondence: Ralf Kircheis, igeneon AG, Immunotherapy of Cancer, Brunnerstrasse 69/3, A-1230 Vienna, Austria. Tel.: +43-1-90 250 205. Fax: +43-1-90 250 901. E-mail:
[email protected].
likely that in different tumors slightly different predominant expression patterns exist, and coexpression of
monomeric and clustered forms (13-15) as well as
heteroclusters consisting of different types of carbohydrates are likely (18).
Our strategy consists of coupling tumor-associated
carbohydrate epitopes to a highly immunogenic murine
antibody with intrinsic antitumor activity (19) in order
to (i) use an immunogenic carrier protein to increase the
immunogenicity of the carbohydrate antigen and (ii) to
benefit from the antitumor immune response induced by
the carrier molecule itself (20, 21). IGN402 is a first
candidate of this type of conjugate vaccine consisting of
SialylTn carbohydrate epitopes chemically coupled to
mAb17-1A. The murine 17-1A antibody, a monoclonal
antibody recognizing EpCAM (19-21), adsorbed on aluminum hydroxide has been used as vaccine antigen in
the cancer vaccine candidate IGN101 and has recently
been reported to prolong survival in metastatic colorectal
cancer patients (22). Recently we have shown that the
SialylTn carbohydrate conjugate vaccine IGN402, coformulated with QS-21 adjuvant, can induce an IgG response against the SialylTn moiety (23).
In the present study we investigated the effect of the
SialylTn coupling density on the immune response.
Furthermore, different types of presentation of SialylTn
(monomeric vs trimers vs clustered via a branched
polyethylenimine linker) were studied. High-density
coupling of SialylTn epitopes to mAb17-1A induced the
strongest immune response against synthetic SialylTn
as well as against natural targets such as OSM and
tumor cells. IGN402 may provide an attractive prototype
for further development toward multi-epitope vaccines
with multiple carbohydrate epitopes coupled to an immunogenic murine antibody.
10.1021/bc050157m CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/01/2005
1520 Bioconjugate Chem., Vol. 16, No. 6, 2005
EXPERIMENTAL PROCEDURES
Coupling of SialylTn Carbohydrate to mAb17-1A
at Different Ratios. The SialylTn carbohydrate antigen
was coupled to the murine monoclonal antibody 17-1A
(mIgG2a) at different molar ratios (i.e. 4.5:1; 18:1; and
36:1) to produce SialylTn-mAb17-1A carbohydrateprotein conjugate vaccines with low, medium, and high
SialylTn density, respectively. Coupling was performed
by reacting 4.5, 18, or 36 mol of nitrophenylated spacered
SialylTn, Neu5AcR2-6GalNAcR-O(CH2)3 NHCO(CH2)4COO-(p-NO2C6H4) (mw 819 g/mol, Lectinity, Finland), to
1 mol of mAb17-1A. Briefly, 70 mg of mAb17-1A was
dialyzed twice against coupling buffer (0.1 M NaPO4, 0.15
M NaCl, pH 8.5) using a Slide-A-Lyzer dialysis cassette
MWCO 10K (Pierce). Concentration of mAb17-1A was
determined by size exclusion chromatography (SEC) on
a Zorbax GF-250. Dialyzed mAb17-1A was diluted to a
final concentration of 4 mg/mL and stored on ice. The
following amounts were transferred to 15 mL vials
(Falcon): 3.5 mL for IGN402 ‘high SialylTn’, 4.6 mL for
IGN402 ‘medium SialylTn’, and 4.6 mL for IGN402 ‘low
SialylTn’. In parallel, 5 mg of SialylTn-O(CH2)4NHCOOpNp was dissolved in 250 µL of DMF, giving a final
concentration of 20 mg/mL. SialylTn (dissolved in DMF)
was added to ice cold mAb17-1A as follows: 22 µL to
IGN402 (low), 90 µL to IGN402 (medium), and 138 µL
to IGN402 (high). The reaction mixture was rotated at
+4 °C. After 53 h, the reaction was stopped by dialyzing
the reaction mixtures against formulation buffer (1 mM
NaPO4, 0.86% NaCl, pH 6) using Slide-A-Lyzer dialysis
cassette 3.5K (Pierce) at 4 °C. For comparison, uncoupled
mAb17-1A was treated in parallel.
To get a very high carbohydrate density, SialylTn was
coupled to mAb17-1A at a molar ratio of 100:1. Briefly,
20 mg of nitrophenylated spacer SialylTn, Neu5AcR26GalNAcR-O(CH2)3NHCO(CH2)4COO-(p-NO2C6H4), was
dissolved in 500 µL of DMF, and 423 µL of this solution
was added to ice cold, dialyzed mAb17-1A (31 mg in 7.75
mL of coupling buffer). The reaction mixture was incubated 74 h under rotation at +4 °C. The coupling product
was dialyzed twice against formulation buffer.
Coupling of SialylTn Carbohydrate Trimers to
mAb17-1A. The SialylTn carbohydrate antigen trimers
attached to one linker molecule having a nitrophenyl
group, [Neu5AcR2-6GalNAcR-O(CH2)3NHCO(CH2)4COGly3-NHCH2-]3 [(p-NO2C6H4)O-CO(CH2)4CO-Gly3-NHCH2)]1C (mw 3105 g/mol, Lectinity, Finland), was coupled to
the mAb17-1A at a molar ratio of 33:1 (calculation based
on trimers). Five milligrams of [Neu5AcR2-6GalNAcRO(CH2)3NHCO(CH2)4CO-Gly3-NHCH2-]3[(p-NO2C6H4)OCO(CH2)4CO-Gly3-NHCH2-)]1C was dissolved in 300 µL
of DMF and added to 8.1 mg of ice cold, dialyzed mAb171A. The reaction mixture was incubated 72 h under
rotation at +4 °C. The coupling product was dialyzed
twice against formulation buffer.
Mimicing SialylTn Clusters on mAb17-1A by Using a Branched PEI Linker. The coupling procedure
consisted of coupling the branched polyethylenimine
(PEI) linker molecule to mAb17-1A, followed by the
coupling of nitrophenylated spacer SialylTn to the mAb171A-PEI conjugate
Coupling of Branched PEI Linker to mAb17-1A.
Branched PEI (25 kDa, Aldrich) was coupled to mAb171A via the heterobifunctional cross-linker SPDP (Nsuccinimidyl 3-(2-pyridyldithio)propionate, Pierce, Rockford, IL). Briefly, mAb17-1A (2.7 mg, 10 mg/mL) and PEI
(4.9 mg, 1 mg/ml) were dialyzed against PBS using SlideA-Lyzer dialysis cassettes. Concentration was measured
Kircheis et al.
using SEC and ninhydrin assay for mAb17-1A and PEI,
respectively.
Coupling of SPDP to mAb17-1A and PEI, Respectively. mAb17-1A (26 mg, 10 mg/mL in PBS) was coupled
with 65 µL of SPDP (20 mM in DMSO) for 60 min at RT.
PEI (4.2 mg, 1 mg/mL in PBS) was coupled with 65 µL
of SPDP (20 mM in DMSO) for 60 min at RT. The SPDP
conjugates were dialyzed against PBS. PEI-SPDP (4.2
mg in 2.1 mL PBS) was reduced with 55 mg of DTT for
30 min at RT. The reduced PEI-SPDP was dialyed
against PBS.
Reaction of mAb17-1A-SPDP with Reduced PEISPDP. Reduced PEI-SPDP (3.95 mg) and mAb17-1ASPDP (24.4 mg) were reacted at a molar ratio of 1:1 for
24 h at +4 °C. The reaction mixture was dialyzed against
PBS using SpectraPor Float-A-Lyzer (MWCO 60 kDa).
The mAb17-1-PEI coupling product was confirmed by
LDS-PAGE, WesternBlot, BioRAD protein assay, and
ninhydrin assay.
Coupling of SialylTn to mAb17-1A-PEI. Ten milligrams of nitrophenylated spacered SialylTn, Neu5AcR26GalNAcR-O(CH2)3NHCO (CH2)4COO-(p-NO2C6H4), were
dissolved in 200 µL of DMF, and added to ice cold mAb171A-PEI conjugate (18 mg/7.2 mL). The reaction mixture
was incubated 75 h under rotation at +4 °C. The coupling
product was dialyzed twice against formulation buffer.
Analysis of Final Coupling Products. Size Exclusion Chromatography. Concentrations of SialylTn-mAb171A coupling products were quantified by size exclusion
chromatography (SEC) on a ZORBAX GF-250 column in
a Dionex system and on a TSKgel G3000SW column in
a HP1100 system.
IEF, LDS-PAGE, and Western Blots. SialylTn-mAb171A coupling products were analyzed by SERVALYT
PRECOTES horizontal flatbed IEF electrophoresis pH
3-10 (SERVA) followed by Coomassie blue staining
(Invitrogen) and by lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE, NuPAGE Electrophoresis System, Bis-Tris-Gel, 4-12%) under both
nonreducing and reducing conditions (50 mM DTT, 1,4dithiothretiol) followed by SilverXpress staining (Invitrogen).
For Western blot analysis, samples following LDSPAGE were transferred (25 V, 1.1 W, 1.5 h) to Immobilon
membranes (PVDF 0.45 µm, Millipore). Membranes were
blocked with 3% skim milk and stained with rabbit antimouse IgG (H+L)-HRP (1:1000, Zymed) or alternatively
with anti-SialylTn CD175s (IgG1) (10 µg/mL, DAKO) and
rat anti-mouse IgG1-HRP (1:1000, Becton Dickinson).
Quantification of Sialic Acid by the Resorcinol Assay.
Amount of sialic acid in the coupling products was
quantified by the resorcinol-HCl reaction in the presence
of CuSO4, and spectrophotometric measurement of absorbance at 562 nm. Briefly, 0.15 mL of samples and 0.15
mL of resorcinol reagent (2% resorcinol, 2.5 mM CuSO4,
HCl (37%)) were mixed in tightly closed glass vials and
incubated at 100 °C for 1 h. The optical density (OD) of
the developing color was measured by adsorption at 562
nm (Spectrophotometer Ultrospec II, LKB Biochrom). As
a standard, a ‘SialylTn mix’ (consisting of sialic acid (i.e.
N-acetylneuraminic acid) and GalNAc at a molar ratio
1:1), 1.25-25 µg/mL, was used for calibration.
Quantification of Sialic Acid by HPLC. Quantification
of amount of sialic acid in the coupling products was
performed by HPLC analysis and fluorescence detection
of DMB derivatives of sialic acid (DMB ) 1,2-diamino4,5-methylenedioxybenzyl). Briefly, samples were treated
with 2 M acetic acid at 80 °C for 3 h and derivatized with
DMB. Derivatized samples were analyzed by reversed
SialylTn Carbohydrate-Conjugate Vaccine
Figure 1. Characterization of SialylTn-mAb17-1A coupling
products with high, medium, or low SialylTn density by IEF,
LDS-PAGE, and Western Blots. Unconjugated mAb17-1A (lane
2) and SialylTn-mAb17-1A conjugates with high (lane 3),
medium (lane 4), and low SialylTn density (lane 5) were
subjected to LDS-PAGE under reducing conditions followed by
silver staining (A), or blotted and stained with anti-SiaTn
CD175s (mIgG1) and rat-anti-mouse IgG1-HRP (B) or rabbitanti-mouse IgG (H+L)-HRP (D), or subjected to IEF-PAGE
and stained by Coomassie blue (C). Heavy and light chains of
mAb17-1A or conjugated SialylTn-mAb17-1A under reducing
conditions (A, B, D) can be seen.
phase HPLC with fluorescence detection. Quantification
was done by the peak area of N-acetylneuraminic acid.
Synthetic N-acetylneuraminic acid (Sigma), 1.25-25 µg/
mL, was used for calibration.
IGN402 Vaccine Formulations. SialylTn-mAb171A conjugate (500 µg) was adsorbed on 1.67 mg of
aluminum hydroxide in 0.5 mL of formulation buffer (1
mM NaPO4, 0.86% NaCl, pH 6) and coformulated with
100 µg of QS-21 adjuvant (Antigenics Inc., Lexington,
MA).
LAL Assay and Pyrogenicity Test in Rabbits.
Amounts of endotoxin in the vaccine formulations were
estimated by Limulus Amebocyte Lysate-Endochrome
Assay (Charles River Endosafe). None of the final vaccine
formulations contained detectable amounts of endotoxin.
Furthermore, the final vaccine formulations were
subjected to pyrogenicity test by iv application in rabbits.
The formulations used for this study were negative
regarding pyrogenicity testing.
Rhesus Monkey Immunization Study. Safety, tolerability, and immunogenicity of multiple subcutaneous
injections of IGN402 were evaluated in a vaccination
study in Rhesus monkeys. All animal studies were
performed under controlled and documented conditions
in accordance with animal health care standards at
Biotest Ltd., Konarovice, Czech Republic. Each SialylTnmAb17-1A conjugate was tested in groups each consisting
of four Rhesus monkeys (sex and age matched). In a first
comparative study animals were vaccinated with four
Bioconjugate Chem., Vol. 16, No. 6, 2005 1521
Figure 2. Quantification of sialic acid by reverse phase HPLC.
Quantification of amount of sialic acid in the coupling products
was performed by HPLC analysis and fluorescence detection of
DMB derivatives of sialic scid. Derivatized samples were
injected into reversed phase HPLC with fluorescence detection.
Quantification was done by measuring the peak area of Nacetylneuraminic acid (NANA). HPLC profile of SialylTnmAb17-1A conjugate (upper panel) and nonconjugated mAb171A (lower panel) are shown. The arrowhead points to the peak
corresponding to NANA in the sample. Synthetic NANA (Sigma),
1.25-25 µg/mL, was used for calibration.
initial immunizations on days (d) 1, 15, 29, and 57. Blood
samples were taken before (d -15, -11, -6) and after
immunization (d 15, 22, 29, 36, 57; 71 and 85).
In a second Rhesus monkey study, four animals per
group were vaccinated with four initial immunizations
on days (d) 1, 21, 49, and 76. Blood samples were taken
before (d -13 and -6) and after immunization (d 27, 49,
52, 70, 79, and 91). All immunizations were well tolerated
by the animals with no signs of systemic or local toxicity
related to immunization. One animal (no. 83 from the
SialylTn[tri]-mAb17-1A group), however, had to be
euthanized during the course of the study because of
moribund conditions. The pathological and histopathological examinations of the animal showed pathologies
in blood coagulation (thrombus in liver vein, embolus in
lungs) most probably not related to the vaccination.
ELISA for Immune Reactivity against mAb17-1A.
Presera and immune sera were analyzed regarding the
induced immune response against mAb17-1A by ELISA.
Briefly, ELISA plates (F96 Maxisorp, NUNC) were coated
with 10 µg/mL mAb17-1A. ELISA plates were blocked
with 2% HSA in PBS (1 h, 37 °C), and samples were
prediluted in PBS with 0.5% HSA and were incubated
for 1.5 h at 37 °C. A positive control serum with known
reactivity against mAb17-1A was tested in parallel and
used for normalization between different ELISA plates.
For detection, plates were incubated with a sheep antihuman IgG-(γ-chain)-HRP conjugate (1:2000, Chemicon)
for 30 min at 37 °C. Staining with substrate OPD (10
mg OPD dissolved in 25 mL + 10 µL 30% H2O2) was
stopped by adding 50 µL of H2SO4 (30%) and measured
1522 Bioconjugate Chem., Vol. 16, No. 6, 2005
Kircheis et al.
Table 1. Molar Ratios of SialylTn to mAb17-1Aa
molar ratio SialylTn to mAb17-1A
coupling product
resorcinol
HPLC (NANA)
mAb17-1A
SiaTn-mAb17-1A (high)
SiaTn-mAb17-1A (medium)
SiaTn-mAb17-1A (low)
16.2 (0.5)
9.1 (0.4)
2.8 (0.1)
12.9
7.1
1.9
a Quantification of sialic acid in the different SialylTn-mAb171A coupling products were estimated by the resorcinol assay. The
resorcinol assay is based on the resorcinol-HCl reaction in the
presence of CuSO4 and spectrophotometric measurement of absorbance at OD562 nm. Mean and standard deviation (SD) are
shown.
at 492/620 nm. The titer was defined as reciprocal serum
dilution yielding an absorbance of OD ) 1.0 on a titration
curve. Curve fitting was done using GraphPad Prism
program version 4.0.
SialylTn-PAA ELISA. Preserum and immune sera
were analyzed concerning the immune response against
the synthetic SialylTn carbohydrate antigen (coupled to
polyacrylamide) by SialylTn-PAA ELISA. Briefly, ELISA
plates (F96 Maxisorp, NUNC) were coated 10 µg/mL
SialylTn-PAA (Lectinity). ELISA plates were blocked
with PBS containing 2% HSA for 1 h at 37 °C. Serum
samples were prediluted in PBS containing 0.5% HSA
and 5% glucose and incubated for 2 h at 37 °C. A positive
control serum with known reactivity against SialylTn
was used for normalization between different ELISA
plates. For detection, plates were incubated with mouse
anti-human IgM-HRP conjugate (1:2000, SB, Southern
Biotechnology) or sheep anti-human IgG-(γ-chain)-HRP
conjugate (1:2000, Chemicon), respectively, for 30 min
at 37 °C. Staining with substrate OPD (10 mg OPD
dissolved in 25 mL + 10 µL of 30% H2O2) was stopped
by adding 50 µL of H2SO4 (30%) and measured at 492/
620 nm. Titers were defined as the reciprocal of serum
dilutions yielding an absorbance of OD ) 1.0 and OD )
0.5 for IgM and IgG, respectively. Curve fitting was done
using GraphPad Prism program version 4.0.
Ovine Submaxillary Mucin (OSM) ELISA. Presera
and immune sera were analyzed regarding the immune
response to OSM which is a natural substrate highly
expressing SialylTn. Briefly, ELISA plates (F96 Maxisorp, NUNC) were coated with 10 µg/mL OSM (Accurate
Chemical, Westbury, NY). ELISA plates were blocked
with 2% HSA for 1 h at 37 °C, followed next by a washing
step. Samples were prediluted in PBS with 0.5% HSA
and incubated for 2 h at 37 °C. A positive control serum
with known reactivity against OSM was tested in parallel
and used for normalization between different ELISA
plates. For detection, plates were incubated with mouse
anti-human IgM-HRP conjugate (1:2000, SB, Southern
Biotechnology) or sheep anti-human IgG-(γ-chain)-HRP
conjugate (1:2000, Chemicon), respectively, for 30 min
at 37 °C. Staining with substrate OPD (10 mg OPD
dissolved in 25 mL + 10 µL of 30% H2O2) was stopped
by adding 50 µL of H2SO4 (30%) and measured at 492/
620 nm. Titers were defined as reciprocal of serum
dilutions yielding an absorbance of OD ) 1.0 and OD )
0.5 for IgM and IgG, respectively. Curve fitting was done
using GraphPad Prism program version 4.0.
Figure 3. Effect of SialylTn coupling density on immune responses induced against SialylTn, mAb17-1A, and OSM. Rhesus monkeys
were immunized with SialylTn-mAb17-1A plus QS-21 vaccines containing high, medium, or low SialylTn coupling density. Preserum
and immune sera at a time kinetic were analyzed for immune response against mAb17-1A carrier protein and SialylTn carbohydrate
antigen and ovine submaxillary mucin by ELISA. Antibody titers (geomean and Scatter factor) against SialylTn (IgG) (A), IgM (B),
mAb17-1A (IgG) (C), and OSM (IgM) (D) are shown. Statistics: *p < 0.05 vs preserum (one-tailed, paired t-test).
SialylTn Carbohydrate-Conjugate Vaccine
Bioconjugate Chem., Vol. 16, No. 6, 2005 1523
Figure 4. Different presentation of SialylTn epitope: monomeric; trimers; clustered via branched linker. The following SialylTnmAb17-1A variants with different presentations of SialylTn were designed and tested in a Rhesus monkey study: monomeric
presentation of SialylTn (A) at high and very high coupling ratios (33:1 and 100:1, respectively); SialylTn presentation as trimers
(ratio 33 [tri]:1) (B); clustered presentation of SialylTn on mAb17-1A-PEI (SialylTn coupling ratio 100:1) (C).
Immune Response against Tumor Cells (FACS
analysis). Binding of immune sera to tumor cells was
measured by cell surface staining using a FACScan
(Becton Dickinson). OVCAR-3 human ovary adenocarcinoma cells (ATCC, HTB-161) were incubated with serum
(diluted 1:40 in PBS with 2% FCS) for 2 h on ice. For
detection, a goat F(ab′)2 anti-human IgG (H+L)-PE
conjugate (1:100, Immunotech, Marseille, France) was
used. Mean fluoresence intensities (MFI) values obtained
for binding of preserum were compared to binding of the
corresponding immune serum. For better comparison, the
binding of preserum of each individual animal was set
at 10% positive cells.
Antibody-Dependent Cellular Cytotoxicity, ADCC.
Preserum and immune serum was tested for ADCC
against SialylTn positive OVCAR-3 cells. Human PBMCs
were used as effector cells and incubated with the 51Crlabeled target cells at different E:T ratios, i.e., 60:1 and
20:1 for 14 h. 51Cr -release was measured by a γ-counter.
RESULTS
Effect of SialylTn Coupling Density on Immune
Responses Induced against mAb17-1A, SialylTn,
and OSM. Nitrophenyl-activated SialylTn carbohydrates
were coupled to mAb17-1A at three different molar ratios
of SialylTn to mAb17-1A (4.5:1; 18:1; and 36:1) resulting
in three SialylTn-mAb17-1A conjugate vaccines with
low, medium, and high SialylTn density, respectively.
SialylTn-mAb17-1A coupling products analyzed by LDSPAGE under reducing conditions followed by Silver
staining are shown in Figure 1A. LDS-PAGE showed
an increase in the molecular weight of the heavy chains
(50 kDa) and the light chains (25 kDa) in the coupling
products (lanes 3-5, compared to uncoupled mAb17-1A,
lane 2) indicating that SialylTn has been coupled to both
heavy and light chains of mAb17-1A. Western blot
analysis of the SialylTn-mAb17-1A coupling products
with either anti-SialylTn CD175s (mIgG1)) and rat antimouse IgG1-HRP (Figure 1B) or alternatively with
rabbit anti-mouse IgG (H+L)-HRP (Figure 1D) demonstrated that SialylTn was detectable in the SialylTnmAb17-1A coupling products but not in the mAb17-1A
(Figure 1B vs 1D). The amount of SialylTn detectable in
the coupling product correlated with the coupling ratio
(lane 3: high, lane 4: medium, lane 5: low). Coupling of
the negatively charged SialylTn to mAb17-1A resulted
also in a shift of the isoelectric point (pI) in the coupling
products as shown by IEF (Figure 1C). The pI shift
correlated with the coupling SialylTn ratio (lane 3: high;
lane 4: medium, lane 5: low SialylTn).
The molar ratios of SialylTn to mAb17-1A in the
coupling products were quantified by Recorcinol assay
or HPLC of N-Acetylneuraminic acid (NANA). In the
resorcinol assay the amount of sialic acid (as part of
SialylTn) in the coupling products is quantified by the
resorcinol-HCl reaction. Molar ratios of SialylTn to
mAb17-1A for the high, medium, and low ratio coupling
product as determined by the resorcinol assay are shown
in Table 1. Quantification of amount of sialic acid in the
coupling products was also performed by reverse phase
HPLC followed by fluorescence detection of DMB derivatives of sialic acid. Quantification is based on the peak
area of N-acetylneuraminic acid (Figure 2). Molar ratios
of SialylTn to mAb17-1A for the high, medium, and low
ratio coupling product as determined by HPLC of NANA
correlate well with those estimated by the resorcinol
assay (Table 1).
The SialylTn-mAb17-1A coupling products (i.e. with
final SialylTn to mAb17-1A ratios of 3:1, 9:1, and 16:1,
1524 Bioconjugate Chem., Vol. 16, No. 6, 2005
respectively, as determined by the resorcinol assay) were
adsorbed on aluminum hydroxide (i.e. 500 µg coupling
product coupled to 1.67 mg aluminum hydroxide) and
coformulated with 100 µg QS-21 adjuvant. Addition of
strong adjuvants, such as QS-21, was previously found
to be essential for induction of switching carbohydrate
specific IgM antibodies to the IgG isotype (23).
Rhesus monkeys, four animals per group, were immunized with SialylTn-mAb17-1A vaccines with high,
medium, or low SialylTn coupling density, respectively.
The time kinetics of the immune responses against
mAb17-1A carrier protein, the SialylTn carbohydrate
antigen, and OSM, a natural substrate with high SialylTn expression, were analyzed by ELISA (Figure 3,
Panels A to D). The induced immune responses against
SialylTn (Panel A: IgG, Panel B: IgM) and OSM (Panel
D: IgM) were found to correlate with the density of
coupled SialylTn. Highest responses against SialylTn
(IgM, IgG) and OSM (IgM) were induced at the highest
coupling density (i.e. 16:1), while only low IgG titers
against OSM were found (data not shown). With the low
SialylTn density (i.e. 3:1) vaccine formulation, generally
only marginal reactivity against SialylTn and no reactivity with OSM were found. In contrast, all three vaccines
induced comparable IgG responses against the mAb171A carrier protein (Panel C). The data demonstrate a
clear correlation between SialylTn ligand density and the
efficacy to induce an anti-carbohydrate immune response,
with the highest tested density showing highest efficacy
of immune response against SialylTn or OSM.
Mimic of Clustered Presentation of SialylTn.
Recent data from the literature indicate that mucinderived, truncated carbohydrates, such as Tn or SialylTn,
are recognized by antibodies preferably as clusters on the
surface of tumor cells rather than as single molecule
epitopes (12-17). To address this issue, the following
variants of vaccines with different presentations of SialylTn were designed: monomeric SialylTn (Figure 4A) coupled at a ratio of 33:1 (corresponding to the highest ratio
of the previous experiment) was compared with a conjugate coupled at very high SialylTn to mAb17-1A ratio,
i.e. 100:1. Furthermore, a trimer SialylTn with three
SialylTn moieties attached to one linker, SialylTn[tri],
was coupled to mAb17-1A at a ratio of 33:1 (trimer-tocarrier ratio) (Figure 4B). Finally SialylTn was coupled
to a mAb17-1A-PEI conjugate at a coupling ratio of 100:
1. The branched polyethylenimine linker molecule, PEI
25 kDa, is a polycation with a high density of primary
amino groups, and coupling of SialylTn to the mAb171A-PEI conjugate aimed to mimic highly clustered
SialylTn presentation (Figure 4C). SialylTn-mAb17-1A
coupling products were analyzed using LDS-PAGE
under reducing conditions (Figure 5A). An increase in
molecular weight of the heavy and the light chains was
found in all coupling products. The SialylTn[tri]-mAb171A conjugate (lane 5) showed a peculiar ladder pattern
with several distinct bands for both heavy and light
chains. Western blot analysis with anti-SialylTn CD175s
(IgG1) and rat anti-mouse IgG1-HRP demonstrated that
SialylTn was detectable in all coupling products, but not
in mAb17-1A (Figure 5B). All coupling products showed
a pI shift compared to mAb17-1A (lane 2) due to the
coupling of negatively charged SialylTn. Different to all
other coupling products, the SialylTn-PEI-mAb17-1A
conjugate repeatedly showed a very peculiar IEF pattern
(lane 6) which may be due to superimposing effects of
the negatively charged SialylTn and the positively charged
PEI (Figure 5C).
Kircheis et al.
Figure 5. Characterization of different SialylTn-mAb17-1A
coupling products by IEF, LDS-PAGE, and Western Blots.
Unconjugated mAb17-1A (lane 2) and SialylTn-mAb17-1A
conjugates, monomeric at coupling ratio 33:1 (lane 3), monomeric at coupling ratio 100:1 (lane 4), SialylTn[tri] at coupling
ratio 33:1 (lane 5), and SialylTn-PEI-mAb17-1A (lane 6) were
subjected to LDS-PAGE under reducing conditions followed by
silver staining (A), or blotted and stained with anti-SiaTn
CD175s (mIgG1) x rat-anti-mouse IgG1-HRP (B), or subjected
to IEF-PAGE and stained by Coomassie blue (C). Heavy and
light chains of mAb17-1A or conjugated SialylTn-mAb17-1A
under reducing conditions (A, B) can be seen.
Table 2. Molar Ratio of SialylTn to mAb in Different
Coupling Productsa
coupling product
molar ratio SialylTn to mAb17-1A
(resorcinol)
SialylTn-mAb17-1A 33:1
SialylTn-mAb17-1A 100:1
SialylTn-mAb17-1A 33[tri]:1
SialylTn-mAb17-1A 100:1
19
75
65
27
a Quantification of sialic acid in the different SialylTn-mAb171A coupling products were estimated by the resorcinol assay. The
resorcinol assay is based on the resorcinol-HCl reaction in the
presence of CuSO4 and spectrophotometric measurement of absorbance at OD562 nm.
SialylTn to mAb17-1A ratios in the final coupling
products were quantified by the resorcinol assay, and the
following ratios (calculated on monomeric SialylTn) were
estimated: 19:1, 75:1, 65:1, and 27:1 for the monomeric
SialylTn-mAb17-1A (reacted at 33:1 and 100:1), SialylTn[tri]-mAb17-1A, and SialylTn-PEI-mAb17-1A, respectively (Table 2). While the molar SialylTn to mAb171A ratios in the 100:1 monomeric SialylTn-mAb17-1A
(final ratio 75:1) and SialylTn[tri]-mAb17-1A (final ratio
65:1) showed, as expected, an approximately 3-fold higher
Bioconjugate Chem., Vol. 16, No. 6, 2005 1525
SialylTn Carbohydrate-Conjugate Vaccine
Figure 6. Effect of SialylTn coupling density and presentation on immune responses induced against synthetic SialylTn and ovine
submaxillary mucin. Rhesus monkeys were immunized with the different SialylTn-mAb17-1A vaccines, adsorbed on alhydrogel,
and coformulated with QS-21 adjuvant. Preserum and immune sera at a time kinetic were analyzed for immune response against
SialylTn carbohydrate antigen and OSM by ELISA. Antibody titers (geomean and Scatter factor) against SialylTn (IgM) (A), IgG
(B), and OSM (IgM) (C), (IgG) (D), are shown.
ratio compared to the 33:1 coupled conjugate (final ratio
19:1); a lower ratio (27:1) was measured in the SialylTnPEI-mAb17-1A coupling product. The SialylTn-mAb171A conjugates were adsorbed on aluminum hydroxide,
coformulated with QS-21 adjuvant and tested in a Rhesus
monkey vaccination study. Presera and the corresponding
immune sera were tested for reactivity against SialylTnHSA and OSM by ELISA (Figure 6). The time kinetics
for the immune responses against SialylTn (Panel A:
IgM, Panel B: IgG) and OSM (Panel C: IgM, Panel D:
IgG) are shown. Again, a strong correlation between
density of coupled SialylTn and the immune responses
against both synthetic SialylTn and OSM was found.
Regarding induced titers, the high coupling product of
monomeric SialylTn to mAb17-1A (coupling ratio at 100:
1, i.e. final ratio 75:1) was found superior to all other
coupling products. The SialylTn[[tri]-mAb17-1A and the
SialylTn-PEI-mAb17-1A conjugates were significantly
more efficient than the monomeric SialylTn (33:1, final
19:1), but were inferior when compared to the 75:1
monomeric coupling product. The superior efficacy of the
high density coupling product was particularly dramatic
concerning the response against OSM, a natural substrate with high SialylTn content (Figure 6C,D).
Immune Response against SialylTn Positive Tumor Cells. Finally, the immune response against the
natural target, SialylTn positive tumor cells, was tested.
Preserum and immune sera of Rhesus monkeys immunized with SialylTn-mAb17-1A plus QS-21 were
analyzed for binding to SialylTn positive OVCAR-3 cells
by FACS analysis. Histograms of IgG and IgM binding
to OVCAR-3 cells are shown for the 75:1 high-density
SialylTn-mAb17-1A vaccine (Figure 7A,B). A statistically significant increase in cell binding to OVCAR-3 cells
(both IgG and IgM) was found for the high coupling
product (p < 0.05, paired t-test). A less consistent
reactivity was found with the other coupling products
(data not shown).
Finally, presera and immune sera of animals immunized with the high-density SialylTn-mAb17-1A vaccine were tested for ADCC against SialylTn positive
OVCAR-3 cells. Human PBMCs were used as effector
cells and incubated with the target cells at two E:T ratios,
i.e., 60:1 and 20:1. While different initial levels of lytic
activity were found in the different animals, a clear
increase in ADCC activity was found in the immune sera
of all animals in comparison to the corresponding presera
(Figure 7C).
DISCUSSION
A panel of synthetic cancer vaccine formulations based
on SialylTn carbohydrate antigens chemically coupled to
an immunogenic protein carrier molecule were designed
and tested in Rhesus monkeys. Coupling of the SialylTn
carbohydrate epitopes to mAb17-1A aims to increase the
immunogenicity of the SialylTn carbohydrate antigen and
to provide with the mAb17-1A carrier an additional
antigen that is thought to mediate an immune response
against epithelial cancer. The murine 17-1A antibody, a
monoclonal antibody (mAb) recognizing the epithelial cell
1526 Bioconjugate Chem., Vol. 16, No. 6, 2005
Figure 7. Rhesus monkeys were immunized with the SialylTn-mAb17-1A vaccines. Preserum (grey line filled) and
immune sera (bold blank line) (both diluted 1:40 in PBS + 2%
FCS) were analyzed for cell binding by FACS analysis (dotted
line: unstained control). Cell binding to SialylTn positive
OVCAR-3 tumor cells is shown for immunization with the highdensity SialylTn coupling product (IgM (A), IgG (B)). Preserum
and immune serum was tested for ADCC against SialylTn
positive OVCAR-3 cells. Human PBMCs were used as effector
cells and incubated with the 51Cr-labeled target cells at E:T ratio
of 60:1 and 20:1 for 14h. 51Cr -release was measured using a
γ-counter (C).
adhesion molecule (EpCAM), has been used for passive
cancer therapy in patients with epithelial carcinomas (19)
whereby part of the observed efficacy has been attributed
to the induction of anti-idiotypic and anti-anti-idiotypic
antibodies (20, 21). In the cancer vaccine candidate
IGN101 mAb17-1A adsorbed on aluminum hydroxide is
being used as vaccine antigen to induce an immune
response against epithelial cancer. IGN101 has demonstrated an excellent safety profile and has recently been
Kircheis et al.
shown to improve survival in metastatic colorectal cancer
patients (22).
In the present study, we investigated the effect of
various SialylTn coupling densities and different ways
of carbohydrate presentation on the carrier molecule on
the induced immune response. Recent data have indicated that the monosaccharide Tn or the disaccharide
SialylTn are predominantly present as molecular clusters
on the surface of tumor cells (9, 12, 17). Therefore,
increasing the coupling density of SialylTn on the carrier
molecule and mimicking various types of clustered
presentation was tested in this study. Our results
demonstrate that at least for this type of carbohydrateprotein conjugate vaccine, the coupling density of SialylTn is an essential parameter determining the efficacy
of the induced immune response against the carbohydrate antigen. While only marginal anti-SialylTn responses were generated at a very low carbohydrate to
mAb17-1A carrier ratio of 3:1, a ratio of ∼16:1 resulted
in significant anti-SialylTn IgG titers. At the highest
coupling density achieved in this study of 75:1, the
highest IgG titers against SialylTn were induced. In
contrast, the immune response against mAb17-1A was
not significantly affected by the SialylTn to mAb17-1A
ratio. However, the magnitude of the immune response
against OSM was again strongly dependent on the
SialylTn coupling density in the vaccine. In particular
IgG antibodies (indicative for an isotype switch from IgM
to IgG) specific for OSM were found at significant levels
only with high SialylTn coupling densities in the vaccine.
Induction of a significant IgG response against OSM and
tumor cells indicates that SialylTn in its natural presentation is also recognized by the induced immune response, although to a lesser degree than the synthetic
SialylTn immunogen. Alternatively designed clustered
presentation of SialylTn as trimers or on a branched PEI
linker did not further improve the induced immune
response against SialylTn, OSM, or tumor cells compared
to the highest monomeric SialylTn coupling density. This
result was somewhat unexpected as trimeric presentation
of SialylTn on an immunogenic carrier has been shown
by others to be a better mimic of the natural presentation
(9, 12, 17). One explanation may be that the recognition
of the trimer SialylTn in the present study is negatively
influenced by the use of longer and more flexible linker
molecules in the trimer conjugate, thus perhaps placing
the three SialylTn moieties at an unfavorably large
distance to each other for recognition. Furthermore,
recent data indicate that it is not only the clustered
carbohydrate but also the context with the peptide anchor
which can affect the immune response (16, 17). It is
currently still under investigation whether the native
mucin glycopeptide architecture (16) or rather the more
immunogenic non-native carbohydrate-peptide linkage
(17) would be better suited for induction of therapeutic
efficacy in tumor patients. Finally, it is known that
different tumor cell lines, depending on the growth
conditions, but also primary tumors and metastatic cells
can differ in their carbohydrate clusters. A trimeric
cluster may be typical for certain cells; for others different
types of clusters may prevail (13, 14, 18).
On the basis of its amino acid sequence, the mAb171A carrier, a murine monoclonal IgG2a antibody, contains a total of 98 lysine residues which present potential
primary amino groups for coupling, depending on the
accessibility. Lysines, due to their positive charge and
extended, rather rigid structure, are generally surface
exposed (25). To get insight in the three-dimensional
distribution of the lysine residues within the murine
SialylTn Carbohydrate-Conjugate Vaccine
Figure 8. Spatial distribution of lysine residues in a 3D-model
of muIgG2a The three-dimensional distribution of the lysine
residues within the murine IgG2a molecule (anti-canine lymphoma mAb 321 (26)) was obtained from the Brookhaven
Protein Data Bank (code: 1IGT), and for visualization the
Rasmol computer software version 2.7.1.1 (ftp://ftp.dcs.ed.ac.uk/
pub/rasmol) was used. The lysines, shown in yellow and light
blue for the two heavy chains, and in green and dark blue for
the two light chains, respectively, appear to be dispersed over
the whole antibody molecule (Panel A: overview on the muIgG2a
molecule in “space-fill” mode). Panel B: a cluster of four lysines
is shown at higher magnification (lysines in ball-stick mode; all
other amino acids in space-fill mode).
IgG2a molecule, the published structure of the anticanine lymphoma mAb 321 (26) was chosen as a model
system. Atomic coordinates for mAb 321 were obtained
from the Brookhaven Protein Data Bank (code: 1IGT),
and for visualization, the Rasmol computer software
version 2.7.1.1 (ftp://ftp.dcs.ed.ac.uk/pub/rasmol) was used.
According to this model (Figure 8, Panel A), the majority
of these lysines (shown in yellow and light blue for the
two heavy chains, and in green and dark blue for the two
light chains, respectively) appear to be dispersed over the
whole antibody molecule (shown in the “space-fill” mode).
In contrast, some of them, especially in the Fc part of
the molecule, are present in spatial proximity to other
lysine residues thus forming clusters (Figure 8, Panel B).
As the free amino groups on accessible lysine side chains
are used for coupling of the sugar moieties to the protein
carrier, clustered lysines may allow for a clustered
coupling and subsequent presentation of carbohydrates.
It should be stressed here that, although mAb321
(muIgG2a) serves only as a model for the mAb17-1A
(muIgG2a) used as the carrier molecule in the present
study, still, based on the high homology, we believe that
this approach is justified. Alignment of the amino acid
Bioconjugate Chem., Vol. 16, No. 6, 2005 1527
sequence of the heavy chain constant regions reveals
100% homology whereas for the variable regions the
homology is, as expected, lower due to the unique
complementary determining regions.
Coupling of carbohydrate epitopes to mAb17-1A may
provide the advantage of creating different types of
presentation for the carbohydrate epitopes, both monomeric as well as different kinds of clusters. Furthermore,
the data of the present study demonstrate that it is
feasible to design a synthetic cancer vaccine which is
adjustable to a controlled and reproducible, large scale
manufacturing process. The SialylTn-mAb17-1A conjugate vaccine may provide a prototype for development of
vaccines with several different carbohydrate epitopes
coupled to the same immunogenic carrier molecule. The
need for high density coupling may limit the number of
different types of carbohydrate epitopes which can be
coupled to the carrier. However, immune recognition of
clustered carbohydrates has been shown so far only for
a selected group of carbohydrates such as Tn and SialylTn and is probably not typical for recognition of larger
carbohydrates, such as GM2 and GloboH. Therefore,
combining carbohydrate epitopes which are recognized
as monomers with epitopes with clustered presentation
on one carrier molecule should be feasible (18). Alternatively, using a mixture of different types of carbohydratecarrier conjugates coformulated in one vaccine formulation might be another pragmatic solution for the generation of a sufficiently broad panel of tumor-associated
carbohydrate recognizing IgG antibodies. Strategies for
cancer vaccines which target multiple tumor antigens
(18, 24) can be expected to provide higher therapeutic
efficacy and to minimize the risk for tumor escape, two
key parameters for the successful treatment of cancer
patients.
ACKNOWLEDGMENT
We thank Dr. C. R. Kensil from Antigenics Inc.,
Lexington, MA, for kindly providing the QS-21 adjuvant
and for critical review of the manuscript.
LITERATURE CITED
(1) Fung, P. Y. S., Madej, M., Koganty, R. R., and Longenecker,
B. M. (1990) Active specific immunotherapy of a murine
mammary adenocarcinoma using a synthetic tumor-associated glycoconjugate. Cancer Res. 50, 4308-4314.
(2) Singhal, A., Fohn, M., and Hakomori, S-I. (1991) Induction
of a R-N-acetylgalactosamine-O-serine/threonine (Tn) antigenmediated cellular immune response for active immunotherapy
in mice. Cancer Res. 51, 1406-1411.
(3) Zhang, S., Walberg, L. A., Helling, F., Ragupathi, G., Adluri,
S., Lloyd, K. O., and Livingston P. O. (1996) Augmenting the
immunogenicity of synthetic MUC-1 vaccines in mice. Cancer
Res. 55, 3364-3368.
(4) Zhang, H., Zhang, S., Cheung, N. K., Ragupathi, G., and
Livingston, P. O. (1998) Antibodies can eradicate cancer
micrometastases. Cancer Res. 58, 2844-2849.
(5) Livingston, P. O., Wong, G. Y. C., Adluri, S., Tao, Y.,
Padavan, M., Parente, R., Hanlon, C., Helling, F., Ritter, G.,
Oettgen, H. F., and Old, L. J. (1994) Improved survival in
AJCC stage III melanoma patients with GM2 antibodies: a
randomized trial of adjuvant vaccination with GM2 ganglioside. J. Clin. Oncol. 12, 1036-1044.
(6) MacLean, G. D., Reddish, M. A., Koganty, R. R., and
Longenecker, B. M. (1996) Antibodies against mucin-associated SialylTn epitopes correlate with survival of metastatic
adenocarcinoma patients undergoing active specific immunotherapy with synthetic STn vaccine. J. Immunother. 19,
59-68.
(7) Itzkowitz, S. H., Yuan, M., Montgomery, C. K., Kjeldsen,
T., Takahashi, H. K., Bigbee, W. L., and Kim, Y. S. (1989)
1528 Bioconjugate Chem., Vol. 16, No. 6, 2005
Expression of Tn, sialosyl-Tn, and T antigens in human colon
cancer. Cancer Res. 49, 197-204.
(8) Zhang, S., Zhang, S. H., Cordon-Cardo, C., Reuter, V. E.,
Singgal, A. K., Lloyd, K. O., and Livingston, P. O. (1997)
Selection of tumor antigens as targets for immune attack
using immunohistochemistry. II. Blood group-related antigens. Int. J. Cancer. 73, 50-56.
(9) Zhang, S., Walberg, L. A., Ogata, S., Itzkowitz, S. H.,
Koganty, R. R., Reddish, M., Gandhi, S. S., Longenecker, B.
M., Lloyd, K. O., and Livingston, P. O. (1995) Immune sera
and monoclonal antibodies define two configurations for the
sialylTn tumor antigen. Cancer Res. 55, 3364-3368.
(10) Itzkowitz, S. H., Bloom, E. J., Kokal, W. A., Modin, G.,
Hakomori, S., and Kim, Y. S. (1990) Sialosyl-Tn: a novel
mucin antigen associated with prognosis in colorectal cancer
patients. Cancer 66, 1960-1966.
(11) Werther, J. L., Rivera-MacMurray, S., Bruckner, H.,
Tatematsu, M., and Itzkowitz, S. H. (1994) Mucin-associated
sialosyl-Tn antigen expression in gastric cancer correlates
with an adverse outcome. Br. J. Cancer 69, 613-616.
(12) Nakada, H., Numata, Y., Inoue, M., Tanaka, N., Kitagawa,
H., Funakoshi, I., Fukui, S., and Yamashina, I. (1991)
Elucidation of an essential structure recognized by an antiGalNAcR-Ser(Thr) monoclonal antibody (MLS 128). J. Biol.
Chem. 266, 12402-12405.
(13) Tanaka, N., Nakada, H., Inoue, M., and Yamashina, I.
(1999) Binding characteristics of an anti-SiaR2-6GalNAcRSer/Thr (sialylTn) monoclonal antibody (MLS132). Eur. J.
Biochem. 263, 27-32.
(14) Ragupathi, G., Howard, L., Capello, S., Koganthy, R. R.,
Qiu, D., Longenecker, B. M., Reddish, M. A., Lloyd, K. O.,
and Livingston, P. O. (1999) Vaccines prepared with SialylTn
and SialylTn trimers using the 4-(4-maleimidomethyl)cyclohexane-1-carboxyl hydrazide linker group result in optimal
antibody titers against ovine submaxillary mucin and SialylTn-positive tumor cells. Cancer Immunol. Immunother. 48,
1-8.
(15) Kagan, E., Ragupathi, G., Yi, S. S., Reis, C. A., Gildersleeve,
J., Kahne, D., Clausen, H., Danishefsky, S. J., and Livingston,
P. O. (2005) Comparison of antigen constructs and carrier
molecules for augmenting the immunogenicity of the monosaccharide epithelial cancer antigen Tn. Cancer Immunol Immunother. 54, 424-430.
(16) Lo-Man, R., Vichier-Guerre, S., Bay, S., Deriaud, E.,
Cantacuzene, D., and Leclerc, C. (2001) Anti-tumor immunity
provided by a synthetic multiple antigenic glycopeptide
displaying a tri-Tn glycotope. J. Immunol. 166, 2849-2854.
(17) Lo-Man, R., Vichier-Guerre, S., Perraut, R., Deriaud, E.,
Huteau, V., BenMohamed, L., Diop, O. M., Livingston, P. O.,
Bay, S., and Leclerc, C. (2004) A fully synthetic therapeutic
vaccine candidate targeting carcinoma-associated Tn carbo-
Kircheis et al.
hydrate antigen induces tumor-specific antibodies in nonhuman primates. Cancer Res. 64, 4987-4994.
(18) Ragupathi, G., Coltart, D. M., Williams, L. J., Koide, F.,
Kagan, E., Allen, J., Harris, C., Glunz, P. W., Livingston, P.
O., and Danishefsky, S. J. (2002) On the power of chemical
synthesis: Immunological evaluation of models for multiantigenic carbohydrate-based cancer vaccines. Proc. Natl. Acad.
Sci U.S.A. 99, 13699-13704.
(19) Riethmuller, G., Schneider-Gadicke, E., Schlimok, G.,
Schmiegel, W., Raab, R., Hoffken, K., Gruber, R., Pichlmaier,
H., Hirsche, H., and Pichlmayr, R. et al. (1994) Randomized
trial of monoclonal antibody for adjuvant therapy of resected
Dukes’ C colorectal carcinoma. German Cancer Aid 17-1A
Study group. Lancet 343, 1172-1174.
(20) Fagerberg, J., Ragnhammar, P., Liljefors, M., Hjelm, A.
L., Mellstedt, H., and Frodin, J. E. (1996) Humoral antiidiotypic and anti-anti-idiotypic immune response in cancer
patients treated with monoclonal antibody 17-1A. Cancer
Immunol. Immunother. 42, 81-87.
(21) Fagerberg, J., Frodin, J. E., Wigzell, H., and Mellstedt, H.
(1993) Induction of an immune network cascade in cancer
patients treated with monoclonal antibodies (ab1). I. May
induction of ab1-reactive T cells and anti-anti-idiotypic
antibodies (ab3) lead to tumor regression after mAb therapy?
Cancer Immunol. Immunother. 37, 264-270.
(22) Settaf, A., Salzberg, M., Groiss, F., Eller, N., Schuster, M.,
Himmler, G., Kundi, M., Eckert, H., and Loibner, H. (2004)
A randomized placebo-controlled Phase II study with the
cancer vaccine IGN101 in patients with epithelial cancers.
Presented at the iSBTc 19th Annual Meeting, San Franscisco,
Nov 4-7.
(23) Kircheis, R., Vondru, P., Zinöcker, I., Häring, D., Nechansky, A., Loibner, H., Himmler, G., and Mudde, G. C. Immunization of Rhesus monkeys with the conjugate vaccine
IGN402 induces an IgG immune response against carbohydrate and protein antigens, and cancer cells. Vaccine, submitted
(24) Ragupathi, G., Koide, F., Santhyan, N., Kagan, E., Spassova, M., Bornmann, W., Gregor, P., Reis, C. A., Clausen, H.,
Danishefsky, S. J., and Livingston, P. O. (2003) A preclinical
study comparing approaches for augmenting the immunogenicity of a heptavalent KLH-conjugate vaccine against epithelial cancer. Cancer Immunol Immunother. 52, 608-616.
(25) Janin, J., and Wodak, S. (1997) Conformation of amino acid
side-chains in proteins. J. Mol. Biol. 125, 357-386.
(26) Harris, L. J., Larson, S. B., Hasel, K. W., and McPherson,
A. (1997) Refined structure of an intact IgG2a monoclonal
antibody. Biochemistry 36, 1581-1597.
BC050157M